Interactions between translation inhibiting antibiotics and diverse RNA regulatory mechanisms affect drug sensitivity in pathogenic bacteria Summary: RNA regulatory mechanisms leverage the tight linkage between transcription, translation, and RNA secondary structure to regulate gene expression through a variety of mechanisms including intrinsic transcription termination, RNA degradation, and translation efficiency. Furthermore, despite findings that a variety of RNA regulators have significant impacts on pathogenicity and virulence, the prevalence of these mechanisms across diverse bacteria is very different. In reaction to sub-inhibitory antibiotics, including translation inhibiting antibiotics (TIAs), different pathogens display diverse physiological responses, driven by wide-spread gene expression changes, that ultimately contribute to the emergence and maintenance of antibiotic resistance. How antibiotics, especially TIAs, affect different types of RNA regulatory mechanisms and thereby contribute to such responses and drug sensitivity is largely unknown. The objective of this proposal is to assess how subinhibitory TIAs interact with RNA regulators to impact transcript boundaries and RNA folding in two bacterial pathogens (Streptococcus pneumoniae and Acinetobacter baumannii) that display diverse paradigms of RNA regulation. We hypothesize that interactions between subinhibitory TIAs and distinct types of RNA regulatory mechanisms have a significant impact on the pathogen?s physiological response and thus drug sensitivity. The rationale for the proposed research is to generate a generalizable mechanistic connection between the subinhibitory doses of TIAs and their physiological effects. To achieve this objective there are two specific aims: 1) Identify changes to transcript boundaries induced by translation inhibiting antibiotics; and 2) establish mRNA structure changes induced by translation inhibiting antibiotics. These aims will be achieved through the use of innovative new sequencing technologies that reach beyond standard coding-transcriptome analysis to evaluate the impact of translation inhibiting antibiotics on transcript boundaries and RNA secondary structure and accessibility. This proposal addresses a significant question whose answer will not only provide insight into the specific organisms examined here, but will also change how past and future transcriptomic studies in bacterial pathogens are analyzed. The expected outcome of this study is a connection between types of RNA regulatory mechanisms within two important pathogens and the physiological effects of subinhibitory TIAs. Furthermore, this knowledge may link genomic features, such as the presence of the termination protein Rho or RNA chaperone Hfq, with observed responses to subinhibitory TIAs that play a role in drug sensitivity and resistance emergence.